Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Efecto inmunosupresor de Trypanosoma lewisi (Kinetoplastidae) sobre la multiplicación de Toxoplasma gondii (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca

The immunosuppressant effect of T. lewisi (Kinetoplastidae) infection on the multiplication of Toxoplasma gondii (Sarcocystidae) on alveolar and peritoneal macrophages of the white rat. The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression. Rev. Biol. Trop. 57 (1-2): 13-22. Epub 2009 June 30.

El efecto inmunosupresor de la infección de T. lewisi sobre la multiplicación de T. gondii fue comparado en macrófagos peritoneales (MP) y alveolares (MA) de rata. El número de parásitos (taquizoitos) intracelulares (TI) fue contado por microscopía de luz. Los macrófagos alveolares y peritoneales (MP) de animales con 4 días de infección con T. lewisi muestran un aumento significativo en la multiplicación de T. gondii. A diferencia de los MP (que muestran un aumento en la multiplicación de T. gondii al cuarto día de infección con T. lewisi disminuyendo hacia el séptimo día), los MA mantienen un aumento en la multiplicación del parásito desde el cuarto, aumentando hacia el séptimo día de infección. Esta diferencia se puede deber a la ruta de infección utilizada para los experimentos que afectan directamente los MP donde se observa un efecto mayor y más temprano en comparación con los MA aislados de los pulmones, compartimiento afectado cuando la infección se vuelve sistémica entre el cuarto y sexto día de infección. La inmunidad contra T. gondii es similar entre ambas células fagocíticas, pero el tiempo de infección y el compartimiento donde se encuentren las células hace la diferencia en el tiempo de respuesta contra un parásito dado, en nuestro caso T. gondii. No hubo evidencia de que los sobrenadantes de cultivos de macrófagos provenientes de ratas infectadas ni el lisado de tripanosomas indujeran el efecto inmunosupresor.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Vaccination against Murine Toxoplasmosis Using Recombinant Toxoplasma gondii SAG3 Antigen Alone or in Combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.

Vaccination against Murine Toxoplasmosis Using Recombinant Toxoplasma gondii SAG3 Antigen Alone or in Combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.

Effect of trypanosoma lewisi infection on the toxoplasma gondii multiplication in white rat peritoneal macrophages

Peritoneal macrophages (PM) from normal Wistar rats were treated in vitro with peritoneal supernatant or sera, obtained from rats infected with 106 Trypanosoma lewisi trypomastigotes before the infection with Toxoplasma gondii tachyzoites. In this experimental model, Toxoplasma multiplication in PM was increased, as compared to macrophages treated with supernatant or sera from control rats not infected with T. lewisi. This effect was observed only if the active supernatant or sera came from rats infected with the T. lewisi 3 to 6 d before Toxoplasma inoculation. Furthermore, immunosuppressive activity was only detectable after at least 24 h incubation with the supernatant or sera. These results are in accordance with our in vivo previous studies which demonstrated a clear immunosuppressive effect of T. lewisi during T.gondii infection of the remarkably resistant Wistar rats.

Detection of toxoplasma gondii in swine sausages

Con el objeto de estudiar la importancia de la longaniza de cerdo en la epidemiología de la toxoplasmosis, se investigó la presencia de Toxoplasma gondii en 70 muestras comercia-lizadas en Botucatu-SP. Las muestras fueron procesadas por las pruebas de aislamiento en ratones y por la amplificación del ADN por la Reacción en Cadena de la Polimerasa (PCR). Aún que el T. gondii no ha sido aislado de ninguna muestra por la prueba de aislamiento in ratones, 33 (47,14 por ciento) muestras han sido positivas por la PCR. Estos resultados indican que la longaniza de cerdo probablemente tiene poca importancia cómo fuente de infección para la toxoplasmosis humana en la región estudiada. Mientras tanto, el gran número de muestras positivas por la PCR indica que el protozoario puede estar presente, pero es inactivado por la sal usada en el aderezo de las longanizas.

Toxoplasma gondii: ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies

This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (P < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.

Long term maintenance of Toxoplasma gondii (Rh strain) in Vero cell line and use of harvested antigens for immunodiagnosis.

Thirty in vitro serial passages of Toxoplasman gondii cultures in Vero cell line performed once in every five days had a mean increase in parasite count of 74.4 +/- 14.8 times from that of initial counts. Long term cultures in Vero cell line did not alter the virulence of the parasite. The good correlation (r = 0.99) between the IFA titer and ELISA OD values using the parasite antigens from in vitro sources indicates that long term maintenance of T. gondii in culture does not affect significantly the ability to recognize antibodies to surface and soluble antigens. The results also show that soluble antigens containing host cells can be directly used for immunodiagnostic purposes without purification. The in vitro maintenance of T. gondii is safer and cheaper when compared to the in vivo method.

Efecto de la infección con Eimeria falciformis sobre el desarrollo de la Toxoplasmosis en ratón / Effect of Eimeria falciformis infection on the development of toxoplasmosis in mice

Se inoculó ratones blancos con 10**2, 10**3 y 10**4 ooquistes de Eimeria falciformis y luego con 10**1, 10**2, 10**3 o 10**4 ooquistes de Toxoplasma gondii, a diferentes períodos despues del inóculo con el primer parásito. En los animales inoculados con las más altas concentraciones de Toxoplasma no se observó variaciones significativas. Sin embargo, en los animales inoculados con 10**1 y 10**2 ooquistes de este último parásito, hubo algunos efectos importantes debidos a la inoculación previa con E. falciformis. Así animales infectados 30 días antes con este parásito presentaron una sobrevivencia mayor al Toxoplasma y el número de quistes encontrados en el cerebro fue considerablemente menor que en los testigos. Adicionalmente el peso de estos últimos fue menor que los previamente infectados con Eimeria, indicando una menor patología presente debida a una menor infección por Toxoplasma

Obtención de ooquistes de toxoplasma gondii mediante inoculación experimental de gatos / Obtention of toxoplasma oocysts by experimental inoculation in cats

Se describe la obtención de ooquistes de Toxoplasma gondii a partir de heces de gatos experimentalmente infectados con quistes del parásito. Lo observado en 17 animales (15 crías y 2 gatas adultas) indica que la producción de ooquistes depende, fundamentalmente, de factores relacionados con el huésped que recibe el inóculo. Para obtener cantidades adecuadas de ooquistes, se recomienda emplear gatos pequeños (aprox. 1 mes de edad), separados de sus madres y libres de infecciones por T. gondii y de cisto-isosporas